Influence of Nonionic Surfactants on the Bioavailability of

Jan 24, 2002 - Influence of Nonionic Surfactants on the Bioavailability of Hexachlorobenzene for Microbial Reductive Dechlorination. Kurt D. Pennell, ...
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Chapter 27

Influence of Nonionic Surfactants on the Bioavailability of Hexachlorobenzene for Microbial Reductive Dechlorination K u r t D. Pennell, Spyros G . Pavlostathis, Ahmet Karagunduz, and Daniel H. Yeh School of C i v i l and Environmental Engineering, Georgia Institute of Technology, Atlanta GA 30332

Experimental and mathematical modeling studies were performed to evaluate the potential benefits and limitations associated with the use of nonionic surfactants to enhance the microbial transformation of hexachlorobenzene (HCB) by a dechlorinating mixed culture enriched from a contaminated sediment. In general, Tween series surfactants were shown to have little impact on methanogenesis, whereas, polyoxyethylene (POE) alcohols, Triton X-100 and SDS were found to strongly inhibit methanogenesis and H C B dechlorination. Subsequent experiments conducted with Tween 80 illustrated the ability of this surfactant to enhance the solubility of H C B and to reduce the HCB-soil distribution coefficient. Model simulations demonstrated, however, that the aqueous phase mass fraction of H C B was substantially reduced in micellar solutions, which corresponded with observed reductions in H C B dechlorination. These results indicate that the impacts of surfactants on both biological activity and contaminant phase distributions should be evaluated in order to accurately assess the potential for biotransformation of hydrophobic contaminants in the presence of surfactants.

© 2002 American Chemical Society

449

450 Hydrophobic organic compounds (HOCs) are commonly found in contaminated soil and sediment systems. A two-phase pattern of H O C desorption, an initial fast stage (in hours) followed by longer, slow phase (in days), has been observed (e.g., 1-4). The resulting low aqueous-phase concentrations limit the availability of HOCs to indigenous microorganisms for biodégradation. Chlorinated organic compounds, many of which are toxic and carcinogenic, are especially resistant to biodégradation due to stability resulting from chlorine substituents. Prytula and Pavlostathis (4) reported that sediment-bound chlorobenzenes exhibited very low bioavailability, with less than 10% of the contaminant mass was reductively dechlorinated over an incubation period of 183 days. Conclusions from this and other studies indicate that efforts to successfully bioremediate contaminated soils and sediments should include means to increase the bioavailability of the sorbed-phase contaminants. Surfactants have been utilized to enhance the removal of both sorbed-phase HOCs and non-aqueous phase liquids (NAPLs) from subsurface systems by increasing the aqueous-phase concentration of HOCs. For example, Pennell et al. (5) demonstrated that surfactant flushing dramatically improved the recovery of residual dodecane from aquifer materials. The ability of surfactants to increase the aqueous solubility of HOCs results from the tendency of surfactants to aggregate in solution to form micelles (6). Surfactants are amphiphilic compounds, possessing both hydrophobic and hydrophilic moieties. A t low concentrations, surfactants exist as monomers in solution and usually exhibit minimal effects on the solubility of HOCs (7). A s the concentration of surfactant in solution is increased, however, the hydrophobic moieties of the monomers tend to associate with one another, eventually forming micelles in solutions. The concentration at which monomer aggregation occurs is referred to as the critical micelle concentration (CMC). It should be noted that, as the surfactant concentration is increased above the C M C the number of monomers in solution remains constant, while the number of micelles continues to increase. In aqueous solutions, surfactant micelles consist of a hydrophobic core surrounded by a hydrophilic mantle. The observed capacity of surfactant solutions to increase the apparent aqueous solubility of HOCs is attributed to the incorporation or partitioning of HOCs within the hydrophobic core of micelles (e.g., 7, 8). This general behavior is depicted in Figure 1, which illustrates a linear increase i n H O C aqueous solubility above the C M C . This relationship can be expressed as the weight solubilization ratio (WSR); WSR =

C ORG

c surf

-C m

OR8,CMC

-c surf,cmc

where C is the concentration of organic species in the aqueous phase ( M / L ) , C is the concentration of the organic species at the C M C (M/L ), C is the 3

org

3

orgcmc

surf

451

Figure 1. Relationship between surfactant concentration and H O C solubility. concentration of surfactant in solution (M/L ), and C is the concentration of surfactant at the C M C (M/L ). A number of researchers have evaluated the use of surfactants to facilitate bioremediation for both in situ and ex situ applications. However, these studies have yielded mixed results, showing either enhanced, reduced or no effect on biodégradation following surfactant addition (9). Such conflicting data can be attributed, in large part, to the numerous interactions that can occur between microorganisms, surfactant monomers and micelles, the solid phase, and the H O C , as depicted in Figure 2. Surfactant monomers can accumulate at the solid-liquid interface and partition into the solid phase (step 1). The latter process may cause swelling of the organic and clay fractions of the solid phase, thereby increasing the rate of contaminant diffusion within the solid matrix (1). In addition to sorption by the solid-phase, surfactant monomers can also sorb to biomass (step 2). It has been hypothesized that the association of surfactants with cell membranes may facilitate mass transfer of HOCs across the membrane, thus enhancing biotransformation (10). The H O C may also participate in sorption processes associated with the solid phase and sorbed surfactant (step 3). A s noted above, partitioning or incorporation of the H O C into surfactant micelles leads to enhanced contaminant solubility (step 4). In most cases, the exchange of HOCs between the aqueous phase and surfactant micelles is considered to be rapid relative to H O C desorption from the solid phase (1,11). Finally, HOCs will tend to accumulate on biomass due to the lipophilic nature of cell membranes. Under proper conditions, the H O C will then be transported into the cell and transformed (step 5). 3

3

surfcmc

452

Figure 2. Conceptual model of coupled interactions among HOCs and the solid phase, biomass and surfactant. To date, the majority of surfactant-enhanced bioremediation studies have focused on aerobic, rather than anaerobic, processes. Thus, the research described herein was specifically designed to evaluate the influence of surfactants on the availability of hexachlorobenzene (HCB) for microbial reductive dechlorination. Hexachlorobenzene was selected for study due to its widespread occurrence in sediments (i2,13), low aqueous solubility, and tendency to be strongly sorbed by soils and sediments. In addition, H C B is classified as a probable human carcinogen by the U.S. Environmental Protection Agency (EPA), and is known to accumulate in the food chain (14). Several classes of nonionic surfactants were evaluated, as well as a representative anionic surfactant. Although this research involved a large matrix of batch, column and modeling studies, the intent of this chapter is to present representative results and to highlight important findings. Specific topics that will be addressed include: a) the impact of surfactants on the methanogenesis and reductive dechlorination of H C B ; b) the influence of surfactants on the aqueous solubility and phase distribution of H C B ; and c) the development and evaluation of mathematical models to describe coupled processes governing H C B bioavailability.

453

Materials and Methods Materials A total of sixteen surfactants were evaluated. Fourteen of the surfactants represented two main classes of nonionic surfactants; linear polyoxyethylene (POE) alcohols (Brij 30, and 35; Witconol SN-70, 90, and 120), and food grade P O E sorbitan esters (Tween 20, 21,40,60, 61, 65, 80, 81, and 85). Within each class, the selected surfactants represent variations in carbon chain length and type, number of ethylene oxide (EO) groups and hydrophile-lipophile balance (HLB). In addition, a representative octylphenol ethoxylate (Triton X-100) and an anionic surfactant, sodium dodecyl sulfate (SDS), were also included in surfactant screening tests. The surfactants were obtained from Aldrich Chemical Co. (Milwaukee, WI) with the exception of Tween 21, 61, 65, and 81, which were obtained from ICI Americas, Inc. (Wilmington, D E ) and the Witconol S N series surfactants, which were obtained from Witco Corporation (Houston, T X ) . The surfactants were used as received from the suppliers without purification. Selected surfactant properties are given in Table I. Surfactant solutions were prepared in deionized, distilled water that had been treated with a Barnstead Nanopure II filter system. Hexachlorobenzene (99+% purity) was obtained from Aldrich Chemical Co. (Milwaukee, WI). Hexachlorobenzene (C C1 ) has a molecular weight of284.8 g/mole, an aqueous solubility of 5μg/L at 25°C (15) and a logarithmic octanolwater partition coefficient (Log K o J of 5.73 (16). A n HCB-contaminated sediment was collected from the Bayou d'Inde, a tributary of the Calcasieu River near Lake Charles, Louisiana. The sediment was used as the inoculum for the HCB-dechlorinating enrichment cultures, as well as one of the media for the examination of the effect of surfactants on H C B desorption and bioavailability. A natural soil (Appling) was also used in batch sorption experiments. Appling soil is classified as a loamy coarse sand of the Appling series (clayey, kaolinitic thermic Typic Hapludult). The organic carbon content of the Appling soil is 7.54 g/kg, and the specific surface area is 3.5 m /g based on nitrogen adsorption. The HCB-dechlorinating culture was developed using anaerobic media and the contaminated sediment as inoculum. The resulting mixed, methanogenic culture was maintained in 9-L sealed glass reactors (6-L liquid volume) with an average hydraulic retention time of 84 days. At each feeding cycle (7-9 days), glucose (333 mg/L), yeast extract (17 mg/L) and H C B (0.025 mg/L) were added to the culture medium. The culture was maintained at an oxidation-reduction potential (ORP) of -330 m V and a p H range of 6.9-7.1. Details of the culture development and maintenance procedures are provided by Yeh et al. (18, 19). 6

6

2

454 Table I. Selected properties of surfactants evaluated in this study Surfactant

Structure"*

MW (g/mole)

CMC (mg/L)

d

HLB

1 1

a

ThOOf (gO/g)

Ρolyoxyethylene (POE) Sorbitan Fatty Acid Esters Tween 20

Ci S EO

20

Tween 21

C S E0

4

Tween 40

1226

44-58

16.7

1.92

522

13

13.3

2.05

C 16^6^020

1282

30-51

15.6

1.98

Tween 60

Cl S EO

20

1310

26-55

14.9

Tween 61

C S E0

4

606

32

9.6

2.24

Tween 65

3(C )S EO

1842

46

10.5

2.34

Tween 80

Cl S EO

20

1308

33-45

15.0

Tween 81

C S E0

5

648

72

10.0

2.20

Tween 85

(3C )S EO

1836

40

11.0

2.32

2

6

12

6

8

6

18

6

18

8

6

6

18

6

18

6

20

20

67

110

2.02

2.01

Ρolyoxyethylene (POE) Alcohols Brij30

C E0

4

362

7-14

9.7

120

2.48

Brij35

Ci E0

23

1198

70-110

16.9

53

2.02

Witconol SN-70

Qo-i2E0

5

392

25

11.2

129

2.36

Witconol SN-90

Cl0-12BO

6

436

36

12.1

118

2.31

Witconol SN-120

C10-12EO9

568

54

13.9

107

2.20

110-150

13.5

140

2.19

12

2

Alkvlphenol Ethoxvlate and Anionic Surfactant Triton X-100

C (pE0 8

SDS

624

95

C OSO^Na

288

12

a

b

40.0

1.81

c

Sorbitan ring (S ). Phenolic ring (φ). Critical micelle concentration, obtained from the supplier and Rosen (6 ). Hydrophile-lipophile balance, calculated as HLB=%wt EO/5 (6). aggregation number (8). 'Theoretical oxygen demand, calculated using method of Metcalf and Eddy, Inc. (17). 6

d

Experimental Methods The first phase of biotic screening was designed to assess the impact of surfactant additions on methanogenic activity of the mixed culture. A serum botde assay was conducted for all sixteen surfactants at an initial surfactant concentration of200 mg/L. Two experimental systems were prepared: 1) surfactant plus glucose,

455 to assess the effect of surfactants on methanogenesis; and 2) surfactant only, to assess the anaerobic biodegradability of the surfactants when present as the only carbon source. Over the 82-day incubation period, total gas and methane production from the surfactant-amended series were measured from the bottle headspace and compared to those from a reference series (containing glucose and no surfactant) and a seed blank series (no carbon source added). The second phase of biotic screening evaluated the effect of Tween surfactants on the reductive dechlorination of H C B by the mixed culture. Experiments were conducted in 28-mL serum tubes sealed with Teflon-lined rubber septa and aluminum crimps. Each tube contained H C B (-140 /xg/L) dissolved in methanol, glucose, and Tween surfactant at concentrations of either 10, 50, 200, or 1,000 mg/L. A t each sampling time, the contents of the tubes were extracted with isooctane followed by an analysis for H C B , dechlorination products and excess gas production. The following parameters were measured: total gas and methane production, pH, ORP, particulate organic carbon, volatile fatty acids (VFAs), and total chemical oxygen demand (COD). The solubility of H C B in aqueous solutions of Tween 60, Tween 80 and Triton X-100 was measured in completely mixed batch reactors over a surfactant concentration range of 0 to 2,000 mg/L. Each 35-mL glass reactor contained excess H C B which was deposited as a thin film on the bottom of the reactor. Analysis of H C B in aqueous surfactant solutions was achieved using a direct injection gas chromatography technique (S). This analytical method yielded more accurate and reproducible results than conventional solvent extraction methods, which resulted in the formation of persistent macroemulsions between the organic solvent and aqueous phases. Batch sorption experiments were conducted using Tween 80 and Appling soil. The batch reactors consisted of either 25-mL glass centrifuge tubes or 30-mL polypropylene centrifuge tubes. The initial concentration of surfactant solutions added to each reactor ranged from 100 to 2,500 mg/L, and contained 0.005 M C a C l as a background electrolyte and 500 mg/L N a N to minimize biological activity. The contents of the reactors were mixed for periods ranging from 1 day to 4 weeks in order to assess adsorption rates and to establish equilibrium sorption capacities. Following mixing, the solid phase was separated by centrifugation (3,000 rpm X 45 minutes) and the resulting supernatant was analyzed for surfactant using a high pressure liquid chromatography (HPLC) system equipped with a diode array detector (DAD) and an evaporative light scattering detector (ELSD). Sorption of H C B by Appling soil was measured in the absence and presence of surfactant. The H C B sorption experiments were conducted in 25 m L Corex glass centrifuge tubes sealed with aluminum-lined caps. The aqueous phase contained 0.005 M C a C l as a background electrolyte and 500 mg/L N a N to minimize biological activity. The effect of Tween 80 on H C B sorption by Appling soil was evaluated as a function of surfactant concentration and mixing time. 2

3

2

3

456 Tween 80 and H C B were added simultaneously, and for each surfactant concentration, the initial concentration of H C B was also varied to allow for the determination of individual H C B sorption isotherms as a function of surfactant loading. In these experiments, the final aqueous phase concentrations of both H C B and Tween 80 were measured independently by G C and H P L C analysis, respectively. Individual and coupled H C B and Tween 80 sorption experiments were conducted in triplicate, with duplicate reference blanks (no solid phase).

Results and Discussion Effect of Surfactants on Methanogenesis The effects of surfactant additions on the rate and extent of electron donor utilization were evaluated based on methane production in the presence and absence of surfactant. Methane production profiles for the mixed culture augmented with 200 mg/L of surfactant and 1,800 mg/L glucose are shown in Figure 3. Although none of the Tween surfactants negatively affected the ultimate extent of methanogenesis compared to the reference series (no surfactant), the presence of Tween 21 did result in a slightly lower initial rate of methanogenesis. In addition, methane production in the presence of Tween surfactants alone was greater than that of the seed blank control, which did not contain electron donor. The linear P O E alcohols, Triton X-100, and SDS greatly inhibited the utilization of glucose for methanogenesis by the mixed-culture (Figure 3). The observed inhibition of methanogenesis in the presence P O E alcohols, which have been used extensively in surfactant enhanced biodégradation studies (9), was not anticipated. Previous studies conducted by Federle and Schwab (20) showed that P O E alcohols were degraded by mixed culture derived from anaerobic sediment. These contrasting results suggest that surfactant-microbial interactions and compatibility are system specific. Effect of Surfactants on Reductive Dechlorination Hexachlorobenzene concentration profiles and total gas production for the glucose-fed culture exposed to three initial surfactant (Tween series) loadings are shown in Figure 4. A t surfactant concentrations of 10 and 200 mg/L, total gas production was identical to that of the reference (without surfactant amendment). As the initial surfactant concentration was increased to 1,000 mg/L (Figure 4C), total gas production was equal to or exceeded the reference case. These data suggest that many of the Tween surfactants were subject to biodégradation. Both the rate and extent of H C B disappearance decreased, however, as the initial surfactant concentration was increased from 10 to 1,000 mg/L. The H C B concentration in azide-amended controls did not vary significantly over the

457

Ο

20

40 60 TIME (Days)

80 Ο

20

40 60 TIME (Days)

80

Figure 3. Methane production by glucose-fed cultures in presence of Tween and non-Tween surfactants at an initial concentration of 200 mg/L; from Yeh et al. (18).

0

20 40 60 TIME (Days)

80

0

5 10 15 20 25 30 35 TIME (Days)

Figure 4. Effects of Tween surfactants on total gas production and H C B disappearance at initial concentrations of 10 (A, D), 200 (Β, E) and 1,000 mg/L (C, F); from Yeh etal. (19).

458 incubation period, indicating that the reduction in H C B concentration was due to microbial activity. A t the lowest Tween surfactant loading (10 mg/L), no change in H C B dechlorination behavior was observed, while at the intermediate loading (200 mg/L) reductions in H C B dechlorination rates were apparent. A t the highest surfactant loading (1,000 mg/L) H C B dechlorination was minimal for all cases except Tween 61 and Tween 65. In summary, H C B dechlorination in the presence of Tween surfactants never exceeded that of the reference (Figure 4D-F), and was, in most cases, greatly reduced at the highest surfactant loading. However, the fact that methane production (Figure 3) and total gas production (Figure 4A-C) were maintained, and in some cases exceeded the reference at the highest surfactant loading, indicate that the presence of Tween surfactants did not directly inhibit methanogenesis, as was the case for the non-Tween surfactants. Nevertheless, the observed reductions in H C B dechlorination suggest that the Tween surfactants altered the biological activity of the mixed culture, either through (a) inhibition or toxicity toward H C B degraders or (b) preferential or competitive utilization of surfactant as a substrate. In addition, the possibility exists that the fraction of H C B incorporated within surfactant micelles was not directly available to the microbial population. The latter hypothesis will be explored in more detail in subsequent sections of this chapter. H C B Solubilization Detailed H C B solubility studies were performed with three surfactants (Tween 60, Tween 80 and Triton X-100) to provide for a range in surfactant properties and biological compatibility. In the absence of surfactant, the solubility of H C B at 25°C was found to be approximately 7 μg/L, which is similar to values reported in the literature (15). Relationships between surfactant concentration and the apparent solubility of H C B are shown in Figure 5. Interestingly, the aqueous-phase concentration of H C B increased by approximately one-order-of-magnitude as the C M C of each surfactant was approached. Similar trends were reported by Kile and Chiou (7), who observed an increase in the apparent solubility of D D T from 5.5 μg/Lin pure water to approximately 70-80 μg/L at the C M C of three Triton series surfactants. This behavior is attributed to interactions between the dissolved H O C and the hydrophobic moiety of surfactant monomers. Above the C M C of each surfactant, linear enhancements in H C B solubility were observed, similar to trends reported for HOCs in micellar solutions (7,8,15). The corresponding W S R values for Tween 60, Tween 80 and Triton X-100, calculated using Equation 1, were 0.59 g/kg, 0.63 g/kg and 0.35 g/kg, respectively. The lower H C B solubilization capacity of Triton X-100 is consistent with solubility correlations developed by Pennell et al. (8) for a range of surfactants and HOCs. This behavior is attributed to the greater alkyl chain length, and hence larger micelle size of Tween 60 and Tween 80 relative to that of Triton X-100.

459

Figure 5. Aqueous solubility of H C B as a function of Tween 60, Tween 80 and Triton X-100 concentration. Surfactant Sorption A matrix of surfactant sorption experiments was conducted as a function of mixing time, initial concentration, and soil type for Tween 60, Tween 80 and Triton X-100. Although only data for Tween 80 and Appling soil are presented here, all of the soil-surfactant systems investigated exhibited Langmuir-type sorption behavior. The Langmuir equation may be written as: S bC 5s = — — s

(2)

K )

l+bC

s

where S is the amount of surfactant sorbed (MM), S is the maximum or limiting sorption capacity (M/M), b is the Langmuir sorption parameter representing the ratio of the adsorption and desorption rates (L /M), and C is the equilibrium surfactant concentration in solution (M/L ). Sorption isotherms for Tween 80 and Appling soil at mixing times of 1 day, 3 days and 7 days are shown in Figure 6. To obtain values of S and è, sorption data were fit to the Langmuir equation using a nonlinear, least-squares regression procedure ( S Y S T A T 5.0). Although the observed values of b were essentially the same after 1, 3 and 7 days of mixing (0.008 L/mg), the maximum or limiting sorption capacity (S ) increased substantially, from 1.44 mg/g to 5.99 mg/g. These data indicate that sorption of s

m

3

s

3

m

m

460 8 • 1 Day • 3 Days • 7 Days A

0 0

200

400 600 Aqueous Tween 80 Cone. (mg/L)

800

1000

Figure 6. Sorption of Tween 80 by Appling soil as a function of mixing time. Tween 80 by Appling soil was strongly rate limited. Based on a theoretical crosssectional molecular area of 300 A /molecule for Tween 80, calculated following the approach of Adeel and Luthy (21), and the specific surface area of Appling soil (3.47 m /g), the limiting sorption capacities were expressed in terms of surface coverage. This analysis yielded Tween 80 surface coverages of 0.6, 1.4 and 2.9 monolayer equivalents after 1,3 and 7 days of mixing, respectively. These results suggest that surfactant loadings corresponding to monolayer surface coverage were achieved within 48 hours, while the formation of surfactant bilayers or hemicelles on the surface, and interactions between Tween 80 and soil organic matter, occurred over longer time frames. 2

2

Coupled Surfactant and HCB Sorption The sorption of H C B by Appling soil was evaluated as a function of surfactant (Tween 80) concentration and mixing time. Tween 80 was selected for these experiments because it readily dissolves in water and remains in solution. Although several of the Tween surfactants (i.e., Tween 60, 61 and 65) were less inhibitory toward H C B dechlorination (Figure 4D-F), these surfactants were more difficult to dissolve in water and were susceptible to the formation of separate phases (e.g., precipitate). The formation of surfactant-rich phases by Tween 60 has also been reported by Shaiu et al. (22). In the absence of surfactant, H C B sorption by Appling soil yielded linear sorption isotherms, with the observed distribution coefficient (K ) increasing from 385 L/kg to 527 L/kg for mixing periods of 1,3,15, and 22 days. In the presence D

461 of surfactant, the initial concentration of H C B was varied to allow for the determination of individual H C B sorption isotherms. The addition of Tween 80 at aqueous phase concentrations above the C M C dramatically reduced the sorption of H C B by Appling soil (Figure 7). As the surfactant concentration was increased, the observed H C B distribution coefficient (K ) decreased substantially. For a given surfactant loading, the H C B distribution coefficient increased with time, indicating that H C B sorption was rate-limited in the presence of surfactant. Since the aqueous phase concentration of Tween 80 also decreased with mixing time due to sorption, these results illustrate the importance of considering the effects of multiple rate limitations on the distribution of hydrophobic organic compounds between the aqueous, micellar and solid phases. D

0

50

100

150

200

250

Aqueous HCB Concentration fog/L)

Figure 7. Hexachlorobenzene sorption isotherms as a function of Tween 80 concentration after 7 days of mixing. Mathematical Modeling of H C B and Surfactant Phase Distributions The overall or apparent solubility of an H O C in the presence of surfactant can be represented as the amount of H O C associated with surfactant monomers plus the amount associated with surfactant micelles. This relationship can be expressed in the following form (23): C* —

=l + C K +C K

r*
"···...

/

/

0

yx #

/

·...

200

400

600

800

1000

Aqueous Tween 80 Cone. (mg/L)

Figure 9. Simulated distribution of H C B among surfactant monomers, micelles, solid phase, and aqueous (free water) phase.

0

200

400

600

800

1000

Aqueous Tween 80 Cone. (mg/L)

Figure 10. Simulation distribution of H C B among surfactant monomers, micelles and aqueous (free water) phase in the absence of soil.

465

Conclusions Results of surfactant biological compatibility studies indicate that methane and total gas production in the presence of Tween series surfactants was similar to, and in some cases exceeded that of, the reference standard (no surfactant). In contrast, P O E alcohols, Triton X-100 and SDS were shown to markedly reduce methane production and to inhibit H C B dechlorination. Within the Tween series, substantial variations in H C B dechlorinating activity were also observed despite similar molecular structures. These results suggest that surfactant compatibility with the HCB-degrading mixed culture was highly surfactant specific. Although methanogenesis was not adversely impacted by the Tween surfactants, rates of H C B disappearance were sequentially reduced as the Tween surfactant concentration was increased from 0 to 1,000 mg/L. These results initially suggested that a) H C B dechlorinating activity of the mixed culture was altered by the Tween surfactants or b) the fraction of H C B available to the microbial population was reduced in micellar solutions. To investigate the latter hypothesis, detailed solubilization, sorption, and H C B phase distribution experiments were performed. Experimental data obtained from these studies showed that Tween 80 enhanced the apparent solubility of H C B both above and below the C M C , Tween 80 was strongly sorbed by a natural soil, and that the HCB-soil distribution coefficient was reduced substantially as the concentration of Tween 80 was increased above the C M C . A mathematical model, developed to simulate H C B phase distributions in the presence of surfactants, clearly demonstrated that the amount of H C B present in the aqueous (water) phase decreased dramatically in the presence of Tween 80. These findings suggest that although Tween 80 was not toxic or inhibitory toward the mixed culture with respect to methanogenesis, micellar solubilization of H C B greatly reduced the mass of H C B available for reductive dechlorination.

Acknowledgements This research was supported by the U.S. Environmental Protection Agency, National Center for Environmental Research and Quality Assurance (Contract No. R-825404-01-0). The contents of this publication has not been subject to agency review, and does not necessarily represent the view of the agency.

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